Physiologically, cells experience and respond to a variety of mechanical stimuli such as rigidity and topography of the extracellular matrix. However, little is known about the effects of substrate curvature on cell behavior. We developed a novel, to our knowledge, method to fabricate cell culture substrates with semicylindrical grooves of negative curvatures (radius of curvature, Rc= 20-100 μm). We found that negative substrate curvatures induced elongation of mesenchymal and epithelial cells along the cylinder axis. As Rcdecreases, mesenchymal National Institutes of Health 3T3 fibroblasts increasingly elongate along the long axis of the grooves, whereas elongation of epithelial Madin-Darby Canine Kidney (MDCK) cells is biphasic with maximal cell elongation when Rc= 40 μm. Addition of blebbistatin to MDCK cells to reduce cortical actin rigidity resulted in a decrease in cell elongation across all curvatures while preserving the biphasic trend. However, addition of calyculin A or ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid, to increase cortical rigidity or reduce intercellular adhesion, respectively, resulted in a monotonic increase in MDCK cell elongation with decreasing Rc. Using an energy minimization model, we showed that cell elongation in epithelial cell sheet is governed by the competition between two energies as Rcdecreases: curvature-dependent intercellular adhesion that prevents elongation; and intracellular cortical actin bending that enhances elongation. Therefore, our results of cellular elongation induced by negatively curved substrates offer insights into how tubule elongation or growth of tubular structures such as kidney tubules can be controlled by the substrate curvature in vivo.